Manuel Sánchez-Gutiérrez

Decreased in vitro fertility in male rats exposed to fluoride-induced oxidative stress damage and mitochondrial transmembrane potential loss.

Fluorosis, caused by drinking water contamination with inorganic fluoride, is a public health problem in many areas around the world. The aim of the study was to evaluate the effect of environmentally relevant doses of fluoride on in vitro fertilization (IVF) capacity of spermatozoa, and its relationship to spermatozoa mitochondrial transmembrane potential (DeltaPsi(m)). Male Wistar rats were administered at 5 mg fluoride/kg body mass/24 h, or deionized water orally for 8 weeks. We evaluated several spermatozoa parameters in treated and untreated rats: i) standard quality analysis, ii) superoxide dismutase (SOD) activity, iii) the generation of superoxide anion (O(2)(-)), iv) lipid peroxidation concentration, v) ultrastructural analyses of spermatozoa using transmission electron microscopy, vi) DeltaPsi(m), vii) acrosome reaction, and viii) IVF capability. Spermatozoa from fluoride-treated rats exhibited a significant decrease in SOD activity (~33%), accompanied with a significant increase in the generation of O(2)() (~40%), a significant decrease in DeltaPsi(m) (~33%), and a significant increase in lipid peroxidation concentration (~50%), relative to spermatozoa from the control group. Consistent with this finding, spermatozoa from fluoride-treated rats exhibited altered plasmatic membrane. In addition, the percentage of fluoride-treated spermatozoa capable of undergoing the acrosome reaction was decreased relative to control spermatozoa (34 vs. 55%), while the percentage fluoride-treated spermatozoa capable of oocyte fertilization was also significantly lower than the control group (13 vs. 71%). These observations suggest that subchronic exposure to fluoride causes oxidative stress damage and loss of mitochondrial transmembrane potential, resulting in reduced fertility.

Protective Effect of Resveratrol on Biomarkers of Oxidative Stress Induced by Iron/Ascorbate in Mouse Spermatozoa

Resveratrol (RVT) is a polyphenolic compound found mainly in the grape and attributed with various pharmacological properties, among them their antioxidant activity. In the present study, we assess the antioxidant activity of resveratrol on oxidative damage induced by ferrous iron/ascorbate (100 µM/150 µM) in sperm of CD1+ mice. We evaluated several parameters in spermatozoa treated with or without resveratrol: (i) sperm quality analysis; (ii) mitochondrial transmembrane potential (Δψm); (iii) ROS generation; (iv) superoxide dismutase (SOD) activity; (v) glutathione peroxidase (GPX) activity; (vi) lipid peroxidation; (vii) and in vitro fertilization (IVF) capability. Spermatozoa treated with RVT (15 µg/mL) before ferrous iron/ascorbate treatment exhibited: a significant increase in motility (8-fold), a significant increase in viability (2-fold), a significant increase in Δψm (1.15-fold), accompanied with a significant decrease in the generation of ROS (4.96-fold), a significant decrease in GPX activity (1.32-fold), and a significant decrease in lipid peroxidation concentration (10.29-fold) relative to spermatozoa treated with ferrous iron/ascorbate; however, no changes in SOD activity were observed. Finally, spermatozoa treated with RVT before ferrous iron/ascorbate treatment showed a significant increase in oocyte fertilization (1.2-fold), relative to spermatozoa treated with ferrous iron/ascorbate. These results suggest that RVT possesses antioxidant properties that may prevent the deleterious effects produced by oxidative damage on spermatozoa, resulting in the maintenance of fertility.

Methyl-parathion decreases sperm function and fertilization capacity after targeting spermatocytes and maturing spermatozoa

Paternal germline exposure to organophosphorous pesticides (OP) has been associated with reproductive failures and adverse effects in the offspring. Methyl-parathion (Me-Pa), a worldwide-used OP, has reproductive adverse effects and is genotoxic to sperm, possibly via oxidative damage. This study investigated the stages of spermatogenesis susceptible to be targeted by Me-Pa exposure that impact on spermatozoa function and their ability to fertilize. Male mice were exposed to Me-Pa (20 mg/kg bw, i.p.) and spermatozoa from epididymis-vas deferens were collected at 7 or 28 days post-treatment (dpt) to assess the effects on maturing spermatozoa and spermatocytes, respectively. Spermatozoa were examined for DNA damage by nick translation (NT-positive cells) and SCSA (%DFI), lipoperoxidation (LPO) by malondialdehyde production, sperm function by spontaneous- and induced-acrosome reactions (AR), mitochondrial membrane potential (MMP) by using the JC-1 fluorochrome, and fertilization ability by an in vitro assay and in vivo mating. Alterations on DNA integrity (%DFI and NT-positive cells) in spermatozoa collected at 7 and 28 dpt, and decreases in sperm quality and induced-AR were observed; reduced MMP and LPO were observed at 7 dpt only. Negative correlations between LPO and sperm alterations were found. Altered sperm functional parameters evaluated either in vitro or in vivo were associated with reduced fertilization rates at both times. These results show that Me-Pa exposure of maturing spermatozoa and spermatocytes affects many sperm functional parameters that result in a decreased fertilizing capacity. Oxidative stress seems to be a likely mechanism of the detrimental effects of Me-Pa exposure in male germ cells.

Role of ATP-sensitive K+ channels in the antinociception induced by non-steroidal anti-inflammatory drugs in streptozotocin-diabetic and non-diabetic rats

There is evidence that systemic sulfonylureas block diclofenac-induced antinociception in normal rat, suggesting that diclofenac activates ATP-sensitive K(+) channels. However, there is no evidence for the systemic interaction between different non-steroidal anti-inflammatory drugs (NSAIDs) and sulfonylureas in streptozotocin (STZ)-diabetic rats. Therefore, this work was undertaken to determine whether two sulfonylureas, glibenclamide and glipizide, have any effect on the systemic antinociception that is induced by diclofenac (30 mg/kg), lumiracoxib (56 mg/kg), meloxicam (30 mg/kg), metamizol (56 mg/kg) and indomethacin (30 mg/kg) using the non-diabetic and STZ-diabetic rat formalin test. Systemic injections of NSAIDs produced dose-dependent antinociception during the second phase of the test in both non-diabetic and STZ-diabetic rats. Systemic pretreatment with glibenclamide (10 mg/kg) and glipizide (10 mg/kg) blocked diclofenac-induced systemic antinociception in the second phase of the test (P<0.05) in both non-diabetic and STZ-diabetic rats. In contrast, pretreatment with glibenclamide or glipizide did not block lumiracoxib-, meloxicam-, metamizol-, and indomethacin-induced systemic antinociception (P>0.05) in both groups. Results showed that systemic NSAIDs are able to produce antinociception in STZ-diabetic rats. Likewise, data suggest that diclofenac, but not other NSAIDs, activated K(+) channels to induce its systemic antinociceptive effect in the non-diabetic and STZ-diabetic rat formalin test.

Antinociceptive, genotoxic and histopathological study of Heliopsis longipes S.F. Blake in mice

ETHNOPHARMACOLOGICAL RELEVANCE H. longipes S.F. Blake (Asteraceae) is a Mexican plant, whose roots are traditionally used as a condiment, as a mouth anesthetic, and as an antiparasitic. Affinin is the alkamide present in higher amounts in the roots of H. longipes. AIM OF THE STUDY To date, there are no published studies regarding the relation between the analgesic properties, in vivo cytotoxicity, and DNA-damaging potential of H. longipes ethanol extract (HLEE). MATERIALS AND METHODS The HLEE was chromatographically fingerprinted to validate its affinin contents. Biological evaluation was conducted in sets of 6-8 CD1(+) mice. Antinociceptive effect was evaluated using the writhing and hot-plate tests, and mutagenic and cytotoxic effects were evaluated with micronucleous test in CD1(+) mice. For histopathological studies, biological samples from liver, heart, kidneys, spleen, lung, and brain were collected and stained. RESULTS Oral administration of HLEE (3-100 mg/kg) produced a dose-dependent antinociceptive effect in both assays. In micronucleus assay, the variability in the number of micronucleated polychromatic erythrocytes (MNPE) induced, and PE/NE index, the ratio of polychromatic erythrocytes with respect to the number of normochromatic erythrocytes induced by HLEE in the evaluated schedule, were small and nonsignificant. After histopathological results, HLEE showed polioencephalomalacia with 1000 mg/kg dose. CONCLUSIONS This work provides evidence that HLEE exerts analgesic effects, with no genotoxic effects in vivo. These findings would be an important contribution to explain the use of H. longipes root as an effective analgesic in traditional medicine, and to establish for the first time the absence of genotoxic and cytotoxic effects of the root in bioactive doses in vivo.

Exposure to bisphenol A in young adult mice does not alter ovulation but does alter the fertilization ability of oocytes.

Follicle growth culminates in ovulation, which allows for the expulsion of fertilizable oocytes and the formation of corpora lutea. Bisphenol A (BPA) is present in many consumer products, and it has been suggested that BPA impairs ovulation; however, the underlying mechanisms are unknown. Therefore, this study first evaluated whether BPA alters ovulation by affecting folliculogenesis, the number of corpora lutea or eggs shed to the oviduct, ovarian gonadotropin responsiveness, hormone levels, and estrous cyclicity. Because it has been suggested (but not directly confirmed) that BPA exerts toxic effects on the fertilization ability of oocytes, a second aim was to evaluate whether BPA impacts the oocyte fertilization rate using an in vitro fertilization assay and mating. The possible effects on early zygote development were also examined. Young adult female C57BL/6J mice (39 days old) were orally dosed with corn oil (vehicle) or 50 μg/kgbw/day BPA for a period encompassing the first three reproductive cycles (12-15 days). BPA exposure did not alter any parameters related to ovulation. Moreover, BPA exposure reduced the percentage of fertilized oocytes after either in vitro fertilization or mating, but it did not alter the zygotic stages. The data indicate that exposure to the reference dose of BPA does not impact ovulation but that it does influence the oocyte quality in terms of its fertilization ability.

Investigation on the Protective Effects of Cranberry Against the DNA Damage Induced by Benzo[a]pyrene

There are few reports that demonstrate the antigenotoxic potential of cranberries. Although the types of berry fruits consumed worldwide are many, this paper focuses on cranberries that are commonly consumed in Mexico (Vaccinium macrocarpon species). The purpose of the present study is to determine whether cranberry ethanolic extract (CEE) can prevent the DNA damage produced by benzo[a]pyrene (B[a]P) using an in vivo mouse peripheral blood micronucleus assay. The experimental groups were organized as follows: a negative control group (without treatment), a positive group treated with B[a]P (200 mg/kg), a group administered with 800 mg/kg of CEE, and three groups treated with B[a]P and CEE (200, 400, and 800 mg/kg) respectively. The CEE and benzo[a]pyrene were administered orally for a week, on a daily basis. During this period the body weight, the feed intake, and the determination of antigenotoxic potential were quantified. At the end of this period, we continued with the same determinations for one week more (recovery period) but anymore administration of the substances. The animals treated with B[a]P showed a weight increase after the first week of administration. The same phenomenon was observed in the lots combined with B[a]P and CEE (low and medium doses). The dose of 800 mg/kg of CEE showed similar values to the control group at the end of the treatment period. In the second part of the assay, when the substances were not administered, these experimental groups regained their normal weight. The dose of CEE (800 mg/kg) was not genotoxic nor cytotoxic. On the contrary, the B[a]P increases the frequency of micronucleated normochromatic erythrocytes (MNNE) and reduces the rate of polychromatic erythrocytes (PE) at the end of the treatment period. With respect to the combined lots, a significant decrease in the MN rate was observed from the sixth to the eighth day of treatment with the two high doses applied; the highest protection (60%) was obtained with 800 mg/kg of CEE. The same dose showed an anticytotoxic effect which corresponded to an improvement of 62.5% in relation to the animals administered with the B[a]P. In the second period, all groups reached values that have been seen in the control group animals. Our results suggest that the inhibition of clastogenicity of the cranberry ethanolic extract against B[a]P is related to the antioxidant capacity of the combination of phytochemicals present in its chemical composition.

Methamidophos alters sperm function and DNA at different stages of spermatogenesis in mice

Methamidophos (MET) is a highly toxic organophosphate (OP) pesticide that is widely used in developing countries. MET has male reproductive effects, including decreased fertility. We evaluated MET effects on sperm quality, fertilization and DNA integrity, exploring the sensitivity of different stages of spermatogenesis. Adult male mice received MET (3.75 or 5mg/kg-bw/ip/day/4 days) and were euthanized 1, 28 or 45 days post-treatment (dpt) to evaluate METs effects on epididymal maturation, meiosis or mitosis, respectively. Spermatozoa were obtained from the cauda epididymis-vas deferens and were evaluated for sperm quality, acrosome reaction (AR; Coomassie staining), mitochondrial membrane potential (by JC-1), DNA damage (comet assay), oxidative damage (malondialdehyde (MDA) production), in vitro fertilization and protein phosphorylation (immunodetection), and erythrocyte acetylcholinesterase (AChE) activity. At 1-dpt, MET inhibited AChE (43-57%) and increased abnormal cells (6%). While at 28- and 45-dpt, sperm motility and viability were significantly reduced with an increasing MET dose, and abnormal morphology increased at 5mg/kg/day/4 days. MDA and mitochondrial activity were not affected at any dose or time. DNA damage (OTM and %DNA) was observed at 5mg/kg/day/4 days in a time-dependent manner, whereas both parameters were altered in cells from mice exposed to 3.75 mg/kg/day/4 days only at 28-dpt. Depending on the time of collection, initial-, spontaneous- and induced-AR were altered at 5mg/kg/day/4 days, and the fertilization capacity also decreased. Sperm phosphorylation (at serine and tyrosine residues) was observed at all time points. Data suggest that meiosis and mitosis are the more sensitive stages of spermatogenesis for MET reproductive toxicity compared to epididymal maturation.

NADPH oxidase participates in the oxidative damage caused by fluoride in rat spermatozoa. Protective role of α‐tocopherol

Fluorosis, caused by drinking water contaminated with inorganic fluoride, is a public health problem in many areas around the world. The aim of this study was to evaluate oxidative stress in spermatozoa caused by fluoride and NADPH oxidase in relationship to fluoride. Four experimental groups of male Wistar rats were administered with deionized water, NaF, at a dose equivalent to 5 mg fluoride kg−1 per 24 h, NaF plus 20 mg kg−1 per 24 h α‐tocopherol, or α‐tocopherol alone for 60 days. We evaluated several spermatozoa parameters in the four groups: standard quality analysis, superoxide dismutase (SOD) activity, the generation of reactive oxygen species (ROS), NADPH oxidase activity, TBARS formation, ultrastructural analyses of spermatozoa using transmission electron microscopy and in vitro fertilization (IVF) capacity. After 60 days of treatment, urinary excretion of fluoride was not modified by α‐tocopherol. Spermatozoa from fluoride‐treated rats exhibited a significant increase in the generation of ROS, accompanied by a significant increase in NADPH oxidase activity. The increase in ROS generation was significantly diminished by diphenylene iodonium, an inhibitor of NADPH oxidase activity. In contrast, a decrease in the generation of ROS, an increase in SOD activity and the prevention of TBARS formation process were observed in spermatozoa of rats exposed to fluoride plus α‐tocopherol. Finally, α‐tocopherol treatment prevented the IVF incapacity observed in the spermatozoa from fluoride‐treated rats. These results suggest that NADPH oxidase participates in the oxidative stress damage caused by subchronic exposure to fluoride. Copyright

Cytotoxic and Antiproliferative Effect of Tepary Bean Lectins on C33-A, MCF-7, SKNSH, and SW480 Cell Lines

For many years, several studies have been employing lectin from vegetables in order to prove its toxic effect on various cell lines. In this work, we analyzed the cytotoxic, antiproliferative, and post-incubatory effect of pure tepary bean lectins on four lines of malignant cells: C33-A; MCF-7; SKNSH, and SW480. The tests were carried out employing MTT and 3[H]-thymidine assays. The results showed that after 24 h of lectin exposure, the cells lines showed a dose-dependent cytotoxic effect, the effect being higher on MCF-7, while C33-A showed the highest resistance. Cell proliferation studies showed that the toxic effect induced by lectins is higher even when lectins are removed, and in fact, the inhibition of proliferation continues after 48 h. Due to the use of two techniques to analyze the cytotoxic and antiproliferative effect, differences were observed in the results, which can be explained by the fact that one technique is based on metabolic reactions, while the other is based on the 3[H]-thymidine incorporated in DNA by cells under division. These results allow concluding that lectins exert a cytotoxic effect after 24 h of exposure, exhibiting a dose-dependent effect. In some cases, the cytotoxic effect is higher even when the lectins are eliminated, however, in other cases, the cells showed a proliferative effect.